1. Austin Brugger Grade 9 Pittsburgh Central Catholic High School

2.  What are the actual effects of alcohol on a Eukaryotic Cell Model ?

3.  Used in many cell/biochemical investigations  Easy to manipulate and rapidly grows  As a eukaryote, it shares similar biochemistry, cell cycle, and genetics with more advanced organisms  Utilized in this study as a human cell model and as a model of human Eukaryotic symbionts or pathogens

4.  More than 10% of your body mass is due to symbionts and pathogens  Mostly Prokaryotic cells  Some may cause diseases  Also protect the body from foreign invaders  Reinforce immune system, prevent allergies, and produce vitamins  Lie within skin, saliva, and gastrointestinal tracts  Break down abnormally large nutrients

5.  Often made through the process of fermentation  Fermentation is the process by which yeast breaks down sugar into carbon dioxide and alcohol.  Common uses include: Household chemicals, alcoholic beverages, and medical chemicals and disinfectants  Very toxic substance with numerous effects on body

6.  To assess the effects of alcohol on the survivorship of Saccharomyces cerevisiae

7. Null Hypothesis: Ethyl alcohol will not affect the survivorship of Saccharomyces cerevisiae. Alternative Hypothesis: Ethyl alcohol will significantly reduce the survivorship of Saccharomyces cerevisiae.

8.  YEPD agar plates (YEPD media + 1.5% agar)  YEPD media (1% yeast extract, 2% peptone, 2% glucose)  Sterile pipette tips  Micropipettes  Vortex  Incubator  Sidearm flask  Spreading turntable  Spreader bar  Ethanol (Ethyl Alcohol)  Sterile capped test tubes with Sterile distilled water.  Saccharomyces cerevisiae (Yeast) (Obtained from Jones Lab, CMU)  0.22 micron syringe filters + 10mL syringe  Klett Spectrophotometer

9. 1. Yeast was grown overnight in sterile YEPD media. 2. A sample of the overnight culture was added to fresh media in a sterile sidearm flask. 3. The culture was placed in an incubator until a density of 50 Klett spectrophotometer units was reached. This represents a cell density of approximately 10 7 cells/mL. 4. The culture was diluted in sterile dilution fluid to a concentration of approximately 10 5 cells/mL. 5. The selected experimental variables were diluted with sterile dilution fluid to the chosen concentrations of 0%, 0.1%, 1%, and 10% to a total of 9.9 mL. 6. 0.1 mL of yeast was then added to the test tubes, yielding a final volume of 10 mL and a cell density of 10 3 cells/mL per tube.

10. Concentration Chart 0%0.1%1%10% Sterile Dilution Fluid 9.9 mL9.89 mL9.8 mL8.9 mL Microbe0.1 mL Ethyl Alcohol0 mL0.01 mL0.1 mL1 mL Total Volume10 mL 7.The solutions were mixed by vortexing and allowed to sit at room temperature 8.After vortexing to evenly suspend cells, 0.1mL aliquots were removed from the tubes at time 10 minutes and 30 minutes and spread on YEPD agar plates (6 plates per concentration)

11. 9. The plates were then incubated at 30 degrees for 48 hours. 10. The resulting colonies were counted. Each colony is assumed to have arisen from one cell.

12. Amount of Colonies per plate (10 min Exposure) Plate 1Plate 2Plate 3Plate 4Plate 5Plate 6Average 0% Alcohol 294255220278285302272 0.1% Alcohol 196180209210204192198 1% Alcohol 163148158184190197173 10% Alcohol 142140138154140136141

13. Amount of Colonies per plate (30 min Exposure) Plate 1Plate 2Plate 3Plate 4Plate 5Plate 6Average 0% Alcohol 205213167186198200202 0.1% Alcohol 159167158164120148153 1% Alcohol 120143140146138123135 10% Alcohol 11011610111210799107

14. P-Value: 0.00000000166 P-Value: 0.000000781 P-Value: 0.000000781

15. 10 Min Exposure Variable ConcentrationT-ValueInterpretation 0.1% Alcohol6.65 Significant 1% Alcohol8.92 Significant 10% Alcohol11.77 Significant 30 Min Exposure Variable ConcentrationT-ValueInterpretation 0.1% Alcohol3.51 Significant 1% Alcohol5.37 Significant 10% Alcohol8.26 Significant T-Critical = 3.1 Alpha= 0.05

16. Concentration of Alcohol Resulting Number of Colonies

17. Ratio of Survivorship of Variables to Control (10 Min Exposure) 0.1% Alcohol1% Alcohol10% Alcohol Control (0% Alcohol) 72%63%51% Ratios of Survivorship of Variables to Control (30 Min Exposure) 0.1% Alcohol1% Alcohol10% Alcohol Control (0% Alcohol) 74%65%52% Did the duration of exposure have a significant effect?

18. Anova: Single Factor 10 Min Exposure SUMMARY GroupsCountSumAverageVariance 0% Alcohol61634272.3333916.2667 0.1% Alcohol61191198.5132.7 1% Alcohol61040173.3333387.0667 10% Alcohol6850141.666740.66667 ANOVA Source of VariationSSdfMSFP-valueF crit Between Groups55788.46318596.1550.372191.66E-093.098391 Within Groups7383.520369.175 Total63171.9623

19. 10 min Exposure MvMcMSEnT 0.10%198.5272.3333369.175673.8333 11.093166 06 6.6557463 91 1%173.3333272.3333369.175699 11.093166 06 8.9244134 11 10%141.6667272.3333369.1756 130.666 6 11.093166 06 11.779017 75 30 Min Exposure T-Crit for both was 3.1 MvMc 0.10%152.6667186270.4417633.3333 9.4945896 87 3.5107678 26 1%135186270.4417651 9.4945896 87 5.3714801 46 10%107.5186270.4417678.5 9.4945896 87 8.2678664 99

20.  Increased concentrations of alcohol lowered the amount of yeast cells that grew.  Increased exposure did not have a significant effect on the amount of surviving colonies.  Higher concentrations of alcohol, since they are more potent, could also be effective in reducing the amount of yeast colonies that could survive.

21.  Incorrect concentrations of alcohol could have been produced.  Spreader bars couldn’t have been sterilized properly  During the testing, plating could have possibly been unsynchronized.  Only one type of alcohol was used.

22.  Different types of alcohol  More alcoholic concentrations  Experiment on Staphylococcus epidermidis and Escherichia coli populations  Study effects on neurological effects on flora of the body  Perform a Try pan blue exclusion assay to test for dead cells.

23.  http://pubs.niaaa.nih.gov/publications/aa63 /aa63.htm http://pubs.niaaa.nih.gov/publications/aa63 /aa63.htm  http://www.alcohol.vt.edu/Students/alcoholE ffects/brainBody.htm http://www.alcohol.vt.edu/Students/alcoholE ffects/brainBody.htm  http://biology.clc.uc.edu/courses/bio104/cel lresp.htm http://biology.clc.uc.edu/courses/bio104/cel lresp.htm  http://www.anaerobicrespiration.net/general /anaerobic-respiration-fermentation/#more- 32 http://www.anaerobicrespiration.net/general /anaerobic-respiration-fermentation/#more- 32  http://www.healthy.net/scr/article.aspx?Id=7 72 http://www.healthy.net/scr/article.aspx?Id=7 72